ABSTRACT DNA replication is a fundamental process for all organisms to precisely duplicate genetic material prior to cell division. Central to the process is a helicase enzyme that utilizes ATP-hydrolysis to separate base- paired DNA to allow polymerases to gain access to synthesize complementary strands as well as to drive the replication machinery along the DNA. In human and other eukaryotic cells, the helicase engine is the hexameric MCM complex. The mechanisms that MCMs use to operate upon DNA are poorly understood at the molecular level. The proposed research will fill a knowledge gap by providing detailed pictures of MCM proteins interacting with DNA and ATP compounds. These will be studied at the molecular level by a coordinated approach involving structural studies by X-ray crystallography and in vitro methods to study their functions and interactions. A considerable body of preliminary data has been obtained for this project that includes MCM:DNA co-crystal structures, important preliminary diffracting crystals of MCM:ADP, purified proteins, and functional information from SPR studies. PUBLIC HEALTH RELEVANCE: RELEVANCE STATEMENT This project focuses on DNA replication, a fundamental event that is required in all life forms. Defects in the replication machinery lead to genetic instability and potentiates tumorigenesis. The central engine of the replication machinery is the hexameric MCM complex that is loaded onto DNA in a regulated process and ultimately unwinds DNA at the replication fork once replication begins. MCMs and the kinases that activate them are logical targets for anti-cancer therapies, and inhibitors of the Cdc7 kinase that phosphorylates MCMs in order to activate its helicase function are currently in clinical trials. The actual mechanisms used by the MCM complex during assembly, activation, and progression upon DNA are not well understood at the structural level. The goals of the project are to study the interactions of MCMs with DNA and how the MCM complex is transformed by molecules of ATP and the ATPase cycle. The project encompasses structural and biochemical techniques working in tandem to study these important biological questions and will provide fundamental insights into mechanisms of replication in human cells.